Sonoko Furuya

Binding of 125I-endothelin-l to fat-storing cells in rat liver revealed by electron microscopic radioautography

SummaryLocalization of intravenously injected [125I]-endothelin-1 was examined in rat liver by light and electron microscopic radioautography. At 10 min after injection, silver grains were localized along the sinusoidal wall, i.e., mostly on the thin processes of fat-storing cells and sinusoidal endothelial cells, and also on the Kupffer cells and the microvilli of hepatocytes. About 35% of the total silver grains were located on the processes of fat-storing cells at 10 min. The grain density (number of silver grains/cell area) of fat-storing cells was three-fold that of Kupffer cells, and 18-fold that of hepatocytes. At 60 min, 60% of the total grains were observed on the fat-storing cells, though the value of grain density was not changed. Silver grains were internalized into the cytoplasm of fat-storing cells and often associated with multivesicular bodies. In contrast, the grain density of endothelial cells and Kupffer cells decreased with time. These results indicate that hepatic fat-storing cells have a considerable number of endothelin-binding sites, and incorporate bound endothelin into cytoplasm.

Single calcium-activated potassium channel in cultured mammary epithelial cells

The properties of Ca2+-activated K+ channels in mouse mammary epithelial cells in primary culture were studied by the patch-clamp technique. In cell-attached patches, spontaneous channel openings were sometimes observed; the slope conductance of the currents was about served; the slope conductance of the currents was about 12 pS at negative membrane potentials with a physiological solution (152 mM Na+, 5.4 mM K+) in the pipette. External application of A23187, a calcium ionophore, activated this channel. In excised inside-out patches, the channel was activated by increasing the internal Ca2+ concentration (10−7 to 10−6 M). No voltage dependence of the channel activity was observed. Internal Na+ blocked the outward K+ current in a voltage dependent manner and this block led to the non-linear I–V relationship at positive membrane potentials. The channel was blocked by internal Ba2+ (0.1 mM) and tetracthylammonium (TEA+, 20–50 mM). Ba2+ reduced the open probability but not the single channel conductance, whereas TEA+ reduced the single channel conductance. The single channel conductance of this channel, measured from the inward current with a high-K+ solution (150 mM K+) in the pipette, was large (about 40 pS), and showed inward rectification. These results suggest that this channel is different from the usual small conductance Ca2+-activated K+ channels observed in many other cells.

Intracellular calcium responses and shape conversions induced by endothelin in cultured subepithelial fibroblasts of rat duodenal villi

Subepithelial fibroblasts of rat duodenal villi were cultured and the physiological characteristics were studied using fura-2 fluorescence. The intracellular calcium concentration (Cai2+) responded to various substances, i.e., endothelins (ET1 and ET3), substance P, serotonin, angiotensin II, ATP, and bradykinin. The Cai2+responses to ET1 (>0.1 nM) and ET3 (>1 nM) were transient and sometimes followed oscillations that consisted of an initial Ca2+ release from the intracellular store and a sustained Ca2+ influx. Simultaneously with Cai2+measurement, changes in the cell shape were monitored using fluorescence intensity upon 360-nm excitation. Stellate cells (with thick cell body and slender processes), formed as a result of 1 mM dibutyryl(Bt2)-cAMP treatment, began to change immediately after the short-term application of the endothelin and became flat about 20 min later. This process was not affected by the depletion of extracellular Ca2+ or by the treatment with BAPTA acetoxymethyl ester that completely suppressed the Cai2+response. Substance P (>100 nM) increased Cai2+, but did not induce any morphological changes. The conversion of the shape from flat to stellate, induced by Bt2cAMP treatment, was not accompanied by any Cai2+change. BQ-123, a specific blocker of the ETA-type receptor, did not block either Cai2+change or shape conversion at low (100 nM) concentration. The results indicated that shape conversion in subepithelial fibroblasts did not require any Cai2+response. Our findings regarding the characteristics of subepithelial fibroblasts in intestinal villi imply a functional similarity to astrocytes in the brain.

Subepithelial fibroblasts in intestinal villi: roles in intercellular communication.

Ingestion of food and water induces chemical and mechanical signals that trigger peristaltic reflexes in the gut. Intestinal villi are motile, equipped with chemosensors and mechanosensors, and transduce signaling to sensory neurons, but the exact mechanisms have not yet been elucidated. Subepithelial fibroblasts located under the villous epithelium form contractile cellular networks via gap junctions. The networks ensheathe lamina propria and are in close contact with epithelium, neural and capillary networks, smooth muscles, and immune cells. Unique characteristics of subepithelial fibroblasts have been revealed by primary cultures isolated from rat duodenal villi. They include rapid reversal changes in cell shape by cAMP reagents and endothelins, cell shape-dependent mechanosensitivity that induces ATP release as a paracrine mediator, contractile ability, and expression of various receptors for vasoactive and neuroactive substances. Herein, we review these characteristics that play a key role in the villi. They serve as a barrier/sieve, flexible mechanical frame, mechanosensor, and signal transduction machinery in the intestinal villi, which are regulated locally and dynamically by rapid cell shape conversion.

Developmental time courses of Na and Ca spikes in neuroblastoma × glioma hybrid cells

The developmental time course of membrane excitability in neuroblastoma X glioma hybrid cells (NG108-15) was analyzed by measuring the electrical potential at each culture day after dBcAMP treatment. The maximum rate of rises of Na and Ca spikes which reflect their inward currents were measured in appropriate medium respectively. The Ca spike began to appear at 2-3 days in culture and reached its maximum on the fifth day. The Na spike began to appear at 3-4 days in culture and was completed on the seventh day. After then Ca and Na spikes remained constant and did not decrease even on the eighteenth day. Electrical excitability of the hybrid cells, as a whole, developed stepwise and was completed on the seventh day of culture.

Characteristics of cultured subepithelial fibroblasts of rat duodenal villi

Subepithelial fibroblasts, which form a cellular network beneath the epithelium of rat intestinal villi, were cultured, and their morphological characteristics were examined. These fibroblasts, which migrated from epithelium-free villi (isolated primarily from duodenum) were flat in shape after 2 days of culture. The flat cells, each resembling a maple leaf, were rich in bundles of microfilaments, and were connected to each other by a few small gap junctions, ascertained from freeze-fracture and dye-coupling experiments. The morphology of the flat cells changed to a stellate form, with thin processes, within 30 min–1 h of reducing the serum concentration and adding dibutyryl cyclic AMP (dBcAMP), forskolin, and cholera toxin. Upon removal of dBcAMP and addition of fetal calf serum, the cells rapidly reassumed their original flat shape. Cells that were stellate in the presence of dBcAMP reassumed a flat morphology within several minutes after the addition of endothelin-1. This conversion occurred with or without extracellular Ca2+. An accompanying rearrangement of the cytoskeleton during shape conversion was observed by fluoresence immunohistochemistry.

Effect and distribution of intravenously injected 125I-endothelin-1 in rat kidney and lung examined by electron microscopic radioautography

SummaryThe morphological effect of endothelin-1 (ET-1) and the distribution of endothelin-binding sites on the kidney and lung was investigated ultrastructually by intravenous injection of [125I]-ET-1 into rats. About 10% decrease of the diameter of glomeruli was observed at 10 min after the injections of ET-1 or [125I]-ET-1 (1.3–2.4 nmole/kg). When localization of [125I]-ET-1 in the kidney was examined by light and electron microscopic radioautography, silver grains were preferably localized on the fenestrated endothelial cells of glomeruli and peritubular capillary endothelial cells. Some grains were also localized on the interdigitating processes of urinary tubules. Quantitative analysis of silver grains in the glomeruli showed that 83% of grains were located on the fenestrated endothelial cells, 12% on the podocytes of visceral cells, and 5% on mesangial cells at 10 min. After 60 min, 50% of silver grains were incorporated into the cytoplasm of fenestrated endothelial cells. In contrast to glomeruli, silver grains were rare on the arteries and large arterioles. However, a few silver grains were often observed on the smooth muscle cells of small arterioles (8–20 μm in diameter). In the lung, 70% of silver grains were located on the alveolar capillary endothelial cells.These results indicate the abundance of ET receptors on the glomerular fenestrated endothelium, peritubular fenestrated endothelium and alveolar capillary endothelium.

Effect and distribution of intravenously injected 125I-guanylin in rat kidney examined by high-resolution light microscopic radioautography.

Abstract 125I-guanylin was injected intravenously into rats, and their kidney and intestinal tract were processed for light microscopic radioautography using semithin sections to examine the binding sites. Various doses of unlabeled guanylin were also injected to examine the morphological effects of guanylin on the kidney. Dense labeling of silver grains due to 125I-guanylin were observed only in the kidney. In the cortex, silver grains were localized on the luminal side of the proximal tubules at 5–30 min. In the medulla, silver grains appeared at the basal side of the collecting ducts, capillaries and loops of Henle after 5 min. Silver grains then accumulated in the cytoplasm of the collecting ducts after 10 min, and disappeared after 30 min. The cell height of the inner medullary collecting ducts (IMCD) decreased and their luminal spaces increased dose-dependently 5 min after the injection of both labeled and unlabeled guanylin. These structural changes returned to control levels within 30 min. These results indicate a high density localization of guanylin receptors on the luminal surface of proximal tubules in the renal cortex and also rapid excretion of guanylin through the IMCD. The morphological changes of the IMCD suggest a diuretic effect of guanylin.

Characteristics of cultured subepithelial fibroblasts in the rat small intestine. II. Localization and functional analysis of endothelin receptors and cell-shape-independent gap junction permeability.

Subepithelial fibroblasts form a cellular network with gap junctions under the epithelium of the gastrointestinal tract. Previously, we have reported their unique characteristics, such as reversible rapid cell-shape changes from a flat to a stellate configuration induced by dBcAMP and endothelins (ETs), and Ca2+ responses to, for example, ETs, ATP, and substance-P. We have now investigated the subtypes of ET receptors both in the rat small intestine and in primary cultured subepithelial fibroblasts isolated from rat duodenal villi. Their properties were compared between wild-type and endothelin-B-receptor-mutant sl/sl rats. Light- and electron-microscopic immunohistochemistry showed intense ETA immunoreactivity in the subepithelial fibroblasts from the small intestine and colon of both wild-type and sl/sl rats. In culture, immunocytochemistry, reverse transcription/polymerase chain reaction analysis, Ca2+ response measurements, and cell-shape change analysis indicated functional ETA and ETB receptors in the wild-type cells, but only ETA in the sl/sl cells. However, wild-type cells were more sensitive to ET-1 than to ET-3 by about one order of magnitude. ETA seemed to be dominant both in vivo and in vitro. The relationship between cell-shape change and gap junction permeability was examined by fluorescence recovery after photobleaching; the gap junctions were usually open but were blocked by carbenoxolone. Permeability did not change significantly with cell-shape change. This network of differentiated subepithelial fibroblasts may maintain intercellular communication via gap junctions to transduce signals evoked in the local network to the whole network. The cell-shape change of the cells through ETA activation may play an important role as a barrier and for intercellular signaling in the intestinal villi.

Intravenous injection of guanylin induces mucus secretion from goblet cells in rat duodenal crypts

Abstract Guanylin, structurally related to the heat-stable enterotoxin of E. coli, is a 15-amino-acid peptide isolated from rat small intestine. We investigated the morphological effects of an intravenous injection of rat and human guanylin upon the rat intestine. Various doses of rat guanylin were injected intravenously in anesthetized rats. After 5, 10 and 30 min, rats were killed by intracardiac perfusion with aldehyde fixative, and specimens of the intestine were then prepared for light and electron microscopy. Intravenously injected rat guanylin rapidly induced mucus secretion from crypt goblet cells in the duodenum. About half of the crypt goblet cells secreted mucous granules by compound exocytosis within 5 min. The villus goblet cells, in contrast, were not sensitive to guanylin. Goblet cells in the jejunum were less responsive than those in the duodenum. This secretory response was rare in the ileum and colon. Human guanylin produced similar results. The mucus secretion induced by guanylin was inhibited by a prior-injection of atropine, but not hexamethonium. Moreover, guanylin induced intense edema in the mucosa and submucosa of the small intestine 5 min after the injection, which disappeared after 30 min. A prior-injection of atropine did not block the appearance of edema. In conclusion, the intravenous injection of guanylin induces two phenomena related to water movement: (1) compound exocytosis of mucous granules from crypt goblet cells in the rat duodenum and jejunum; (2) perineural, inter-epithelial and intra-epithelial edema in the rat small intestine.